B lymphocytes are selected early in development for further maturation in peripheral sites. This positive selection step involves the emigration of lymphocytes form the bone marrow to secondary lymphoid organs where they enter lymphoid follicles. Initially generated B lymphocytes are short-lived and after two distinguishable maturation steps these lymphocytes receive a signal that converts them into a long-lived form. Bruton's tyrosine kinase (Btk) is functionally linked to the B-cell receptor and is indispensable for the conversion of short-lived B-cells into long-lived cells. The least mature short-lived cells can be distinguished from all other peripheral B-cells by their inability to proliferate in response to antigen receptor ligation. A major goal of these studies is to molecularly dissect the checkpoint that regulates the conversion of short-lived B-cells into long-lived cells. The order of events in peripheral B-cell maturation will be established by following a cohort in in vivo labeled B-cells and also by experiments involving the transfer of purified B-cell populations. Cell death at presumed checkpoints will be assessed in wild type mice and in selected mutant strains. The least mature peripheral B-cell fraction will be functionally compared with immature bone-marrow B-cells (which are believed to be critical for the generation of tolerance). The mechanisms involved in preventing cells at this stage from proliferating in response to antigen receptor signaling will be investigated. Six putative molecular targets of Btk and three molecular targets of Lyn (a Src-family tyrosine kinase associated with the B-cell receptor), all of which are phosphorylated on tyrosine residues after B-cell activation, will be characterized in detail. A complete analysis of targets of these kinases may yield important clues to the mechanisms involved in generating long-lived B-cells and in antigen receptor mediated B-cell proliferation. Since the majority of human lymphomas develop form activated B-cells, these signaling mechanisms are likely to be of direct relevance for a significant proportion of human cancers.